Biological indicators (BIs) are used globally to monitor the efficacy of sterilization processes for medical products (1–5). BIs contain high numbers—generally 104 to 106—of bacterial endo-spores that are highly resistant to the sterilization process for which they are designed. The spores are placed on or in a carrier material such as paper and packaged to protect the BI before as well as after sterilization and before culturing. BIs are useful for validation and routine monitoring of ethylene oxide, steam, dry heat, low temperature steam formaldehyde, vapor hydrogen peroxide, and related plasma sterilization systems.

In the United States, BIs sold for use in healthcare facilities are considered medical devices and are regulated by the US Food and Drug Administration good manufacturing practices (GMPs) as promulgated in the Quality System Regulation of 1996 (6). BIs sold to manufacturers of sterile medical products are considered manufacturing components. Regardless of the intended use, BIs must be manufactured under carefully controlled conditions to assure quality, reproducibility, and predictable performance (7).

Reducing biological indicator incubation time and the existing FDA protocol

How much incubation time is required to provide sufficient information to allow the user to make a correct decision regarding the acceptability of the sterilization process? In the mid-1980s, the FDA Center for Device and Radiological Health (CDRH) issued a protocol (hereafter termed the RIT [reduced incubation time] protocol) for BI manufacturers and users to follow to reduce the incubation time for BIs from the normal seven days to a shorter time based on the results of testing defined in the protocol (8). The RIT protocol requires manufacturers to expose 100 BIs from each of three lots to separate sterilization processes; the results of the exposure to the sterilization processes must result in 30 to 80 nonsterile BIs from each lot after incubation at the appropriate conditions for seven days. Achieving this outcome requires carefully-controlled reduced lethality (fractional) sterilization runs for which highly specialized sterilization equipment is often used.

Table I : Example of data necessary to meet the FDA CDRH criteria of determining a reduced incubation time of less than 7 days.

To determine a reduced incubation time for a BI/sterilization process according to the RIT protocol, the number of nonsterile BIs for each of the three lots is recorded after periodic reviews up to seven days of incubation; the number of nonsterile BIs at seven days is considered "100%." The number of nonsterile BIs at incubation times less than seven days is compared with the respective seven-day value, and a percentage is calculated; the qualified reduced incubation time is the longest time, for one or more of the three lots, to first exhibit a number of nonsterile BIs that is ≥ 97.0% of that observed after seven days of incubation. A typical example appears in Table I with the BIs being scored daily.

As required, for each of the three lots tested, the number of nonsterile BIs after seven days of incubation was greater or equal to 30 and less than or equal to 80. For lots 2 and 3, the 97.0% criterion was met after one day of incubation; for lot 1, however, the 97.0% criterion was met after two days of incubation. Based upon these results, a reduction of incubation time from seven to two days is acceptable.